Piccinini Filippo, Tesei Anna, Arienti Chiara, Bevilacqua Alessandro
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Via Piero Maroncelli 40, 47014 Meldola, FC Italy.
Advanced Research Center on Electronic Systems "Ercole De Castro" (ARCES), University of Bologna, Via Toffano 2/2, 40125 Bologna, Italy.
Biol Proced Online. 2017 Jul 20;19:8. doi: 10.1186/s12575-017-0056-3. eCollection 2017.
Whatever the target of an experiment in cell biology, cell counting and viability assessment are always computed. The Trypan Blue (TB) assay was proposed about a century ago and is still the most widely used method to perform cell viability analysis. Furthermore, the combined use of TB with a haemocytometer is also considered the standard approach to estimate cell population density. There are numerous research articles reporting the use of TB assays to compute cell number and viability of 2D and 3D cultures. However, the literature still lacks studies regarding the reliability of the TB assay in terms of assessment of its repeatability and reproducibility.
We compared the TB assay's measurements obtained by two biologists who analysed 105 different samples in double-blind for a total of 210 counts performed. We measured: () the repeatability of the count performed by the same operator; () the reproducibility of counts performed by the two operators.
There were no significant differences in the results obtained with 2D and 3D cell cultures: we estimated an approximate variability of 5% when the TB assay was used to assess the viability of the culture, and a variability of around 20% when it was used to determine the cell population density.
The main aim of this study was to make researchers aware of potential measurement errors when TB is used with a haemocytometer for counting and viability measurements in 2D and 3D cultures. We believe that these results can help researchers to determine whether the expected reliability of the TB assay is compliant with their applications.
无论细胞生物学实验的目标是什么，细胞计数和活力评估都是常需进行的操作。台盼蓝（TB）检测法大约在一个世纪前被提出，至今仍是进行细胞活力分析最广泛使用的方法。此外，将TB与血细胞计数器结合使用也被视为估计细胞群体密度的标准方法。有大量研究文章报道了使用TB检测法来计算二维和三维培养物中的细胞数量和活力。然而，就TB检测法在评估其重复性和再现性方面的可靠性而言，文献中仍缺乏相关研究。
我们比较了两位生物学家通过双盲方式对105个不同样本进行分析所获得的TB检测法测量结果，总共进行了210次计数。我们测量了：（）同一操作人员计数的重复性；（）两位操作人员计数的再现性。
二维和三维细胞培养所获得的结果没有显著差异：当使用TB检测法评估培养物的活力时，我们估计其变异性约为5%，而当用于确定细胞群体密度时，变异性约为20%。
本研究的主要目的是让研究人员意识到，当在二维和三维培养物中使用TB与血细胞计数器进行计数和活力测量时可能存在的测量误差。我们相信这些结果可以帮助研究人员确定TB检测法的预期可靠性是否符合其应用需求。
